This invention relates to the cloning and expression of foreign proteins and polypeptides in bacteria such as Escherichia coli. In particular, this invention relates to the expression of bacterially synthesised foreign proteins or polypeptides as fused polypeptides which are in general soluble and which can be readily purified from bacterial lysates without altering the antigenicity or destroying the functional activity of the foreign protein or polypeptide.
A number of different vectors have been described that direct the expression of foreign polypeptides in Escherichia coli (reviewed by Harris, 1983; Marston, 1986; McIntyre et. al., 1987). Some vectors have been designed to simplify product purification but a difficulty common to most of these systems is that denaturing reagents are required to maintain the solubility of proteins or to elute them from affinity reagents. Denaturation may be expected to alter the antigenicity and destroy any functional activity of the foreign polypeptide. For example, polypeptides expressed as NH.sub.2 -terminal or COOH-terminal fusions with E. coli .beta.-galactosidase (Gray et. al., 1982; Koenen et. al., 1982; Ruther & Muller-Hill, 1983) and that are soluble in 1.6M NaCl, 10 mM .beta.-mercaptoethanol can be purified from crude cell lysates by affinity chromatography on a column of p-aminophenyl-.beta.-D-thiogalactoside followed by elution in 0.1M sodium borate pH 10, 10 mM .beta.-mercaptoethanol (Germino et. al., 1983; Ullmann, 1984). Such fusion proteins will also bind to immobilised anti-.beta.-galactosidase antibodies and can be recovered by elution in solutions of high or low pH (Promega biotec). Alternatively, fusions with .beta.-galactosidase that lack the last 16 COOH-terminal amino acids of .beta.-galactosidase are frequently insoluble (Itakura et. al., 1977; Young & Davis, 1983; Stanley & Luzio, 1984) and so can be purified from the insoluble fraction of lysed bacteria after resolubilisation by treatment with denaturing reagents (Marston, 1986). The same method can be used to purify polypeptides expressed as insoluble COOH-terminal fusions with a protein containing the trpE leader sequence and the COOH-terminal third of the trpE protein (Kleid et. al., 1981). Apart from the use of denaturing conditions, these methods suffer from the disadvantage that the E. coli proteins used as carriers may dominate an immune response to the fusion protein, particularly in the case of fusions with .beta.-galactosidase (Mr 116,000), and may elicit antibodies that show undesirable cross-reactions.
Other expression vectors direct the synthesis of polypeptides as fusions with the COOH-terminus of staphyloccocal protein A that can be purified from cell lysates by affinity chromatography on a column of human IgG-Sepharose (Uhlen et. al., 1983; Nilsson et. al., 1985; Abrahmsen et. al., 1986; Lowenadler et. al., 1986). Because of the high affinity of protein A for IgG, denaturing conditions are usually reguired for the elution of fusion proteins although alternative strategies can be employed such as competition with excess native protein A, the use of sheep IgG which has a lower affinity for protein A (Nilsson et. al., 1985) or reduction in the size of the protein A carrier such that its affinity for IgG is reduced (Abrahmsen et. al., 1986). A more serious difficulty is that the binding of fusion proteins to IgG complicates the immunological screening of clones or analysis of recombinant products since antibodies that bind to protein A will recognise every fusion protein, regardless of their other specificities.
Another strategy for the purification of foreign polypeptides from E. coli is to produce polypeptides that contain poly-arginine at their COOH-terminus (Sassenfeld & Brewer, 1984). The strongly basic arginine residues increase the affinity of fusion proteins for anionic resins so that fusions can be purified by cation exchange chromatography, following which the COOH-terminal arginine residues can be removed by treatment with carboxypeptidase B. Other vectors direct the secretion of polypeptides into the periplasmic space or into the culture medium and although levels of expression are often low, secreted polypeptides are protected from degradation by bacterial proteases and separated from most other proteins (Marston, 1986; Abrahmsen et. al., 1986; Lowenadler et. al., 1986; Kato et. al., 1987). These last approaches have been used successfully in some instances, but their generality is unclear, particularly for polypeptides containing many acidic residues or that are largely hydrophobic.